Axonemes were isolated from sperm of Colobocentrotus by a procedure involving two extractions with 1% Triton X-100 and washing The isolated axonemes contained 7 X 10 -is g protein per/*m of their length. Treatment of the axonemes with 0 5 M I~CI for 30 min extracted 50-70 % of the flagellar ATPase protein, dynein, and removed preferentially the outer arms from the doubIet tubules. Almost all of the dynein (85-95 %) could be extracted from the axonemes by dialysis at low ionic strength. In both cases the extracted dynein sedimented through sucrose gradients at 12-14S, and no 30S form was observed The enzymic properties of dynein changed when it was extracted from the axonemes into solution. Solubilization had a particularly marked effect on the KC1-and pH-dependence of the ATPase activity. The pH-dependence of soluble dynein was fairly simple with a single peak extending from about pH 6 to pH 10. The pH-dependence of bound dynein was more complex. In 0.1 M KC1, the bound activity appeared to peak at about pH 9, and dropped off rapidly with decreasing pH, reaching almost zero at pH 7; an additional peak at pH 10 0 resulted from the breakdown of the axonemal structure and solubilization of dynein that occurred at about this pH. A similar curve was obtmned m the absence of KC1, except for the presence of a further large peak at pH 8 Measurement of the kinetic parameters of soluble dynein showed that both K=~ and V~x increased with increasing concentrations of KCt up to 0.5 )a When bound dynein was assayed under conditions that would induce motility m reactivated sperm (0 15 )a KC1 with Mg ++ activation), it did not obey Michaelis-Menten kinetics, although it did when assayed under other conditions. The complex enzyme-kinetic behavior of bound dynein, and the differences between its enzymic properties and those of soluble dynem, may result from its interactions with tubulin and other axonemal proteins
A high-resolution sodium dodecyl sulfate polyacrylamide gel electrophoresis system has been used to show the presence, in both whole sperm and isolated flagellar axonemes, of eight polypeptides migrating in the 300,000--350,000 molecular weight range characteristic of the heavy chains of dynein ATPase. Previously, only five such chains have been discernible. Extraction of isolated axonemes for 10 min at 4 degrees C with a solution containing 0.6 M NaCl, ph 7, releases a mixture of particles that separate, in sucrose density gradient centrifugation, into a major peak, dynein 1 ATPase, sedimenting at 21S and a minor peak at 12--14S. The polypeptide compositions of these two peaks are different. The dynein 1 peak, which contains most of the protein on the gradient, contains approximately equal quantities of two closely migrating heavy chains, with a small amount of a third, more slowly migrating chain; no other heavy chains appear in this peak. Two groups of smaller polypeptides (three intermediate chains, within the apparent molecular weight range 76,000--122,000 and four newly discovered light chains, within the apparent molecular weight range 14,000--24,000) cosediment with the 21S peak. The heavy chain composition of the 12--14S peak is more complex, all eight heavy chains occurring approximately the same ratios as occur in intact axonemes.
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